scholarly journals Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells

1970 ◽  
Vol 119 (5) ◽  
pp. 839-848 ◽  
Author(s):  
J. G. Lindsay ◽  
S. Berryman ◽  
R. L. P. Adams
Keyword(s):  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Partial Purification ◽  
Isolated Nuclei ◽  
Exonuclease I ◽  
Mouse Cells ◽  
Dna Template ◽  
L929 Mouse

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2–5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50μg of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg2+ and K+ concentrations. 6. Maximal enzyme activity is approached with 40μg of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The 3H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the 3H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.

scholarly journals Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases

1979 ◽  
Vol 178 (3) ◽  
pp. 621-626 ◽  
Author(s):  
J F Burke ◽  
P M Duff ◽  
C K Pearson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Acid Synthesis ◽  
Cell Nuclei ◽  
Isolated Nuclei ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta ◽  
Deoxyribonucleic Acid Synthesis

In order to ascertain the identity of the DNA-dependent DNA polymerase responsible for the observed DNA synthesis in nuclei isolated from baby-hamster kidney (BHK-21/C13) cells a comparative study was carried out on the effects of some drugs, reported to influence DNA synthesis, on DNA synthesis catalysed by these nuclei and by partially purified DNA polymerase-alpha and -beta. In all cases DNA synthesis by isolated nuclei and polymerase-alpha was inhibited to similar extents by N-ethylmaleimide, p-hydroxymercuribenzoate, novobiocin, heparin and phosphonoacetic acid; polymerase-beta was much less affected by these compounds. Ethidium bromide inhibited all DNA synthesis to similar extents, although at low concentrations (about 2 microgram/ml) synthesis in isolated nuclei was stimulated. The results are discussed in relation to the proposal that DNA polymerase-alpha catalyses the covalent extension of Okazaki fragments that these nuclei carry out in vitro.

THE EFFECTS OF AGE AND OESTRONE TREATMENT ON DNA POLYMERASE ACTIVITY IN ANTERIOR PITUITARY GLANDS OF MALE RATS

1973 ◽  
Vol 59 (1) ◽  
pp. 107-119 ◽  
Author(s):  
ANDREA MASTRO ◽  
W. C. HYMER
Keyword(s):  
Dna Polymerase ◽  
Anterior Pituitary ◽  
Reaction Medium ◽  
Polymerase Activity ◽  
Male Rats ◽  
Synthetic Activity ◽  
Cytoplasmic Fraction ◽  
Isolated Nuclei ◽  
Pituitary Glands

SUMMARY DNA polymerase activity was found in the cytoplasmic fraction and in isolated nuclei from anterior pituitary glands of male rats. The enzyme activity was assayed by measuring the incorporation of [3H]dTTP into DNA in a medium containing Tris-HCl buffer (pH 8·5), the four deoxyribonucleoside triphosphates, Mg2 +, ATP and activated calf thymus DNA. The DNA polymerase activity decreased with age in glands from animals aged 25 days to over a year but increased after oestrone treatment in vivo. These changes in activity, more pronounced in the cytoplasmic fraction than in the isolated nuclei, were similar to changes in DNA synthesis measured in anterior pituitary glands under the same physiological conditions. Isolated nuclei also retained endogenous DNA synthetic activity in the absence of added template. Addition of a cytoplasmic fraction to the reaction medium stimulated activity by as much as 1·9-fold but the degree of stimulation was the same whether the cytoplasm was from young, old or oestrone-treated animals.

scholarly journals Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle

1976 ◽  
Vol 154 (2) ◽  
pp. 387-393 ◽  
Author(s):  
W C. Claycomb
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Cardiac Muscle ◽  
Dna Polymerase ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Neonatal Rat ◽  
Dibutyryl Cyclic Amp ◽  
Fresh Tissue ◽  
Dna Polymerase Α

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase α and thymidine kinase. When DNA synthesis and the activity of DNA polymerase α are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.

scholarly journals Efficient Gap Repair Catalyzed In Vitro by an Intrinsic DNA Polymerase Activity of Human Immunodeficiency Virus Type 1 Integrase

1998 ◽  
Vol 72 (3) ◽  
pp. 2062-2071 ◽  
Author(s):  
Andrea Acel ◽  
Brian E. Udashkin ◽  
Mark A. Wainberg ◽  
Emmanuel A. Faust
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Single Stranded Region ◽  
Dna Template ◽  
Hiv 1

ABSTRACT Cleavage and DNA joining reactions, carried out by human immunodeficiency virus type 1 (HIV-1) integrase, are necessary to effect the covalent insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanking the integrated proviral DNA must be repaired. It has been widely assumed that host cell DNA repair enzymes are involved. Here we report that HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA polymerase activity. The activity was characterized by its dependence on Mg2+, resistance toN-ethylmaleimide, and inhibition by 3′-azido-2′,3′-dideoxythymidine-5′-triphosphate, coumermycin A1, and pyridoxal 5′-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or self-annealing oligonucleotides as a template primer but displayed relatively low activity with gapped calf thymus DNA and no activity with poly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifically to an epitope comprised of amino acids 264 to 273 near the C terminus of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drastically altered the gel filtration properties of the DNA polymerase, although the level of activity was unaffected by this mutation. The DNA polymerase efficiently extended a hairpin DNA primer up to 19 nucleotides on a T20 DNA template, although addition of the last nucleotide occurred infrequently or not at all. The ability of integrase to repair gaps in DNA was also investigated. We designed a series of gapped molecules containing a single-stranded region flanked by a duplex U5 viral arm on one side and by a duplex nonviral arm on the other side. Molecules varied structurally depending on the size of the gap (one, two, five, or seven nucleotides), their content of T’s or C’s in the single-stranded region, whether the CA dinucleotide in the viral arm had been replaced with a nonviral sequence, or whether they contained 5′ AC dinucleotides as unpaired tails. The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently and completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5′-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was removed, leaving a nongapped DNA template-primer, the integrase DNA polymerase failed to repair the last nucleotide in the DNA template effectively. A post-gap repair reaction did depend on the CA dinucleotide. This secondary reaction was highly regulated. Only two nucleotides beyond the gap were synthesized, and these were complementary to and dependent for their synthesis on the CA dinucleotide. We were also able to identify a specific requirement for the C terminus of integrase in the post-gap repair reaction. The results are consistent with a direct role for a heretofore unsuspected DNA polymerase function of HIV-1 integrase in the repair of short gaps flanking proviral DNA integration intermediates that arise during virus infection.

scholarly journals Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in rat liver after protein restriction

1975 ◽  
Vol 148 (1) ◽  
pp. 49-56 ◽  
Author(s):  
G M Andersson ◽  
A von der Decken
Keyword(s):  
Rna Polymerase ◽  
Dietary Protein ◽  
Deoxyribonucleic Acid ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Rna Polymerase I ◽  
High Quality ◽  
Sephadex Column ◽  
High Quality Protein ◽  
Polymerase I

Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from α-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.

Deoxyribonucleic Acid Polymerase of Trout Liver Nuclei

1970 ◽  
Vol 27 (1) ◽  
pp. 117-123 ◽  
Author(s):  
H. L. A. Tarr ◽  
Linda Gardner
Keyword(s):  
Dna Polymerase ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Freezing And Thawing ◽  
Low Concentrations ◽  
Mandatory Requirement ◽  
Mammalian Tissues ◽  
Liver Nuclei ◽  
Deoxynucleoside Triphosphate ◽  
High Concentrations

The DNA polymerase of intact trout liver nuclei behaved similarly to that present in mammalian tissues in its mandatory requirement for four deoxynucleoside triphosphates, Mg2+, and DNA primer. It was inhibited by DNase and pyrophosphate in comparatively low concentrations and by RNase and Actinomycin D in comparatively high concentrations. Soluble DNA polymerase was extracted from buffered suspensions of the nuclei by centrifugation at 100,000 g. It possessed properties very similar to those exhibited by the enzyme in intact nuclei. The polymerase activity of intact nuclei was increased by freezing and thawing, and the soluble enzyme was unstable at 30 C and lost most of its activity in 1 day at 0 C. The specific activity of the enzyme was 10–29 picomoles of deoxynucleoside triphosphate incorporated into DNA by 1 mg of protein in 1 hr at 25 C.

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